Stability

Enzymes are stable only under certain conditions. In their physiological environ-ment they can be active for severals weeks, whereas the isolated enzyme is not stable at room temperature for a long time. As enzymes are proteins, they are very sensitive to changes of temperature and pH value of their surroundings.

The stability of two different enzyme reactors, one with chicken liver sulfite oxidase and one with plant sulfite oxidase, was compared. Both reactors were prepared the same day with approximately the same amount of sulfite oxidase enzyme.

Storage conditions were exactly the same for both enzyme reactors at all times.

The reactors were applied in the analysis of standard sulfite solutions, as well

3.3.ComparisonofSulfiteOxidases R! = 0,99996

R! = 0,88809

0 10 20 30 40 50 60 70

0 0,5 1 1,5 2 2,5 3

Peak area

Concentration of SO₂ in mg/L Plant sulfite oxidase

Animal sulfite oxidase

Figure 3.18.: Calibration curves in the concentration range of 0.04 – 2 mg/L SO2. Comparison between plant and animal sulfite oxidase in HPLC-IMER.

83

3. Results and discussion

as of food samples. Each sample was injected two times consecutively, with the switch valve changing between reactors after each sample. Thus, both reactors were exposed to the same conditions at all times.

In table 3.6, some data of this experiment is presented. Within two months, both reactors have been extensively stressed with many different grape juice samples and standard solutions.

Table 3.6.: Comparison of animal and plant sulfite oxidase.

Area of sulfite standard Day # Injections

Sulfite std. sol. Plant SOx Animal SOx

Ratio

animal/plant SO2

0 1 12.7 9.75 0.77

1 50 11.5 8.9 0.77

6 100 11.2 6.8 0.61

14 150 8.3 2.7 0.33

42 200 12 3.9 0.33

58 250 9.3 1.3 0.14

Figure 3.19 shows the comparison of the obtained peak areas relative to each other. Out of 250 different samples and standard solutions analyzed, only the values for the standard solutions are displayed for reasons of better comparability.

Food samples generally contain a lot more known and unknown substances than a standard solution, which might influence the performance of either one or both of the enzymes and therefore lead to incommensurable results. The results, however, were found to be very similar for food samples.

At the beginning of the experiment, the reactor with animal sulfite oxidase showed an activity level of 77% compared to the plant sulfite oxidase reactor. After 50 injections, the ratio was still about the same. From thereon, the activity of the animal SOx decreased compared to that of the plant sulfite oxidase. After 200 injections, the peak areas of a standard solution with the animal sulfite oxidase reactor were only 34% of that of the plant SOx, after 250 injections the ratio decreased down to 14%.

It is necessary to compare both enzyme reactors relative to each other, as the absolute peak area of a reactor may vary due to different factors (see chapter 3.2).

It is not possible to determine the exact decrease of one sulfite oxidase reactor’s reactivity.

3.3.ComparisonofSulfiteOxidases

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1 50 100 150 200 250

ratio of peak areas

injections

!"#$%&'()&*+,'-.#/%0,'

1&%"+'()&*+,'-.#/%0,' 223'

453'

Figure 3.19.: Comparison of sulfite oxidase performances. Both enzymes were used under the same conditions.

Peak areas of standard solutions are presented, the area of plant sulfite oxidase is set at 100% for better comparison.

85

3. Results and discussion

After two months and 250 injections of standard sulfite solutions and food samples, the performance of the animal sulfite oxidase reactor became too weak for sensitive sulfite analysis with HPLC-IMER. The peak area of a standard solution with 0.4 mg/L SO2 was only 13% of its initial value. In comparison, the plant sulfite oxidase reactor still showed about 73% of its initial peak area after the same amount of time and stress.

From both experiments, it can be concluded that for application in the HPLC-IMER, the plant sulfite oxidase is better suited than the sulfite oxidase from chicken liver.

The broad linear range of the plant sulfite oxidase allows for a one point calibra-tion for all samples within the deteccalibra-tion range of the electrochemical detector.

With the animal sulfite oxidase reactor this is not possible, as the linear range is a lot smaller. One would either have to accept not very exact results, or a calibra-tion curve would be necessary for every sample. The latter is an inefficient and time consuming step. As described earlier, the response of a reactor may change within an hour. Therefore, a calibration curve for every sample appears not at all practical for the HPLC-IMER.

The better immobilization stability of the plant oxidase is a further reason to prefer this enzyme over the animal sulfite oxidase. In most cases, the plant SOx reactor leads to higher responses from the start, and shows a much better stability compared to the animal sulfite oxidase. Some plant SOx reactors were still appli-cable after two years of moderate use, whereas the animal SOx reactors showed either very small peaks or no signals at all after the same period of time.

Conclusively, both sulfite oxidase enzymes are suited for use in the HPLC-IMER.

Compared to the chicken liver SOx, the plant SOx shows two major advantages:

The immobilized plant sulfite oxidase is more stable, and it can be used over a longer period of time than the animal sulfite oxidase. The linear range of the plant sulfite oxidase reactor is a lot bigger than that of the animal sulfite oxidase, limited only by the maximum load of the electrochemical detector.

The application of a marine sulfite oxidase in HPLC-IMER analysis was not suc-cessful. A sulfite detection after immobilization of the marine sulfite oxidase on the carrier material was not achieved.